Field of the Invention
The present invention concerns quantifiable risk factors for the post-exposure development of equine herpesvirus-1 neurological disease and their use to provide a new equine experimental disease model for evaluating and developing effective vaccines for the protection of horses against equine herpesvirus-1 neurological disease. The invention further relates to a new live, attenuated vaccine and its use to prevent neurological disease caused by equine herpesvirus-1.
Description of Related Art
All patents and publications cited in this specification are hereby incorporated by reference in their entirety.
Epizootics of neurological disease caused by equine herpesvirus-1 (EHV-1) have been reported with increasing frequency in the United States during the past several years (United States Department of Agriculture, “Equine herpes virus myeloencephalopathy: A potentially emerging disease,” USDA: APHIS: Veterinary Services: Centers for Epidemiology and Animal Health, Center for Emerging Issues Information Sheet, 2007). Characterized by high neurologic morbidity and case fatality rates, resistance to prevention by vaccination, and the ability to affect horses of all breeds and ages, EHV-1 myeloencephalopathy has the potential for causing catastrophic losses to both the welfare of horses and the economy of equine-based businesses (M. T. Donaldson and C. R. Sweeney, “Equine herpes myeloencephalopathy,” Compend Contin Edu Pract Vet 19:864-871 (1997); W. D. Wilson, “Equine herpesvirus 1 myeloencephalopathy,” Vet Clin North Am Equine Pract 13:53-72 (1997); R. W. Henninger et al., “Epidemic neurologic disease due to equine herpesvirus-1 at a university equestrian center,” J Vet Intern Med 21:157-165 (2007); C. van Maanen et al., “Neurological disease associated with EHV-1 infection in a riding school: clinical and virological characteristics,” Equine Vet J 33:191-196 (2001); M. J. Studdert et al., “Outbreak of equine herpesvirus type 1 myeloencephalitis: new insights from virus identification by PCR and the application of an EHV-1-specific antibody detection ELISA,” Vet Rec 153:417-423 (2003); S. M. Reed and R. E. Toribio, “Equine herpesvirus 1 and 4,” Vet Clin North Am Equine Pract 20:631-642 (2004); T. A. Jackson and J. W. Kendrick, “Paralysis of horses associated with equine herpesvirus 1 infection,” J Amer Vet Med Assoc 158:1351-1357 (1971); K. M. Charlton et al., “Meningoencephalomyelitis in horses associated with equine herpesvirus 1 infection,” Vet Pathol 13:59-68 (1976); P. B. Little and J. Thorsen, “Disseminated necrotizing myeloencephalitis: a herpes-associated neurological disease of horses,” Vet Pathol 13:161-171 (1976); H. Platt et al., “Pathological observations on an outbreak of paralysis in broodmares,” Equine Vet J 12:118-126 (1980); R. S. Greenwood and A. B. Simpson, “Clinical report of a paralytic syndrome affecting stallions, mares and foals on a Thoroughbred studfarm,” Equine Vet J 12:113-117 (1980); K. E. Whitwell and A. S. Blunden, “Pathological findings in horses dying during an outbreak of the paralytic form of Equid herpesvirus type 1 (EHV-1) infection,” Equine Vet J 24:13-19 (1992); C. W. Kohn and W. R. Fenner, “Equine herpes myeloencephalopathy,” Vet Clin N Am: Equine Pract 3:405-419 (1987)).
Although the specific immune mechanisms required for control of EHV-1 neurologic disease are largely not established, a well known immunoeffector mechanism for controlling the level of cell-associated viremia of other herpesviruses is cytotoxic T lymphocytes (CTL) (A. M. Arvin, “Cell-mediated immunity to varicella-zoster virus,” J Infect Dis 166:35-41 (1992); S. Martin et al., “Herpes simplex virus type 1 specific cytotoxic T lymphocytes recognize virus nonstructural proteins,”J Virol 62:2265-2273 (1988)). Later investigations of Kydd et al. demonstrated that resistance to EHV-1 abortion in ponies was associated with high frequencies of pre-exposure, EHV-1 specific CTLp in the blood circulation (J. H. Kydd et al., “Pre-infection frequencies of equine herpesvirus-1 specific, cytotoxic T lymphocytes Fcorrelate with protection against abortion following experimental infection of pregnant mares,” Vet Immunol & Immunopathol 96:207-217 (2003)). However, the Kydd et al. study dealt solely with abortion as a disease outcome of EHV-1 infection and did not address the neurological manifestation of EHV-1 infection.
Recent molecular characterization of EHV-1 isolates recovered from outbreaks of neurologic disease has revealed that the majority of isolates are variant strains of the virus that possess an adenine (A) to guanine (G) base substitution at position 2254 of the gene that encodes the viral DNA polymerase (open reading frame #30; ORF30) (J. Nugent et al., “Analysis of equine herpesvirus type 1 strain variation reveals a point mutation of the DNA polymerase strongly associated with neuropathogenic versus non-neuropathogenic disease outbreaks,” J Virol 80:4047-4060 (2006)). It has been postulated that this unique mutation in the catalytic subunit of the viral replication complex endows such virus strains with an enhanced replicative vigor that increases the number and severity of lesions of necrotizing vasculitis in the blood vessels of the central nervous system of the infected horse (K. E. Whitwell and A. S. Blunden, “Pathological findings in horses dying during an outbreak of the paralytic form of Equid herpesvirus type 1 (EHV-1) infection,” Equine Vet J 24:13-19 (1992); G. P. Allen and C. C. Breadthnach, “Quantification by real-time PCR of the magnitude and duration of leukocyte-associated viraemia in horses infected with neuropathogenic versus non-neuropathogenic strains of equine herpesvirus-1,” Equine Vet J 38:252-257 (2006)).
Concern has been voiced that genetic change in the herpes viral agent with a concomitant increase in its virulence is resulting in the neurological manifestation of EHV-1 infection that is increasing in incidence, morbidity and case fatality rates (United States Department of Agriculture, “Equine herpes virus myeloencephalopathy: A potentially emerging disease,” USDA: APHIS: Veterinary Services: Centers for Epidemiology and Animal Health, Center for Emerging Issues Information Sheet, 2007). The increase in incidence of the high-mortality herpesviral neurological disease of horses that is not protected against by currently marketed vaccines has fueled genuine concerns about its effect on the future economic prosperity of the U.S. horse industry. Of particular alarm to the equine industry is its recent targeting of horses in riding/boarding stables and of young horses assembled at race track venues for training and racing, with consequent high mortality rates and severe economic losses to the boarding and racing sectors of the industry. Although efforts to develop a more effective, second-generation vaccine against the neurologic herpesvirus disease are underway by several vaccine manufacturers, no equine disease model exists for assessing the effectiveness of such experimental vaccines.
In fact, a practical neurological disease model has never been created, either in connection with standard laboratory mice, rats or guinea pigs where the neurological effects of EHV-1 infection can never develop, or even from prior equine studies of EHV-1 in horses or ponies. Earlier challenge studies, including those reported in the published paper by T. O'Neill et al., “Determination of equid herpesvirus 1-specific, CD8+, cytotoxic T lymphocyte precursor frequencies in ponies,” Vet Immunol & Immunopathol 70:43-54 (1999), did not address the neurological manifestation of EHV-1 infection or disease. Since the oldest group of 9-year-old ponies in that 1999 study showed frequencies of EHV-1 specific CTLp that were high before experimental infection, it was concluded by the co-authors that high CTLp frequency may correlate with immunity to EHV-1. Furthermore, the ponies never reached the point where they presented any neurological signs. Their blood analysis and physical characteristics are of limited value, consequently, in determining the factors giving rise to neurological disease, the traits of a neurological disease model or the criteria useful in predicting development of neurological disease.
Previously, vaccination strategies have focused on the well-known murine model of EHV-1 infection in which the efficacy and safety of the EHV-1 vaccines are initially tested in vivo in mice rather than directly in horses (see, for example, A. R. Frampton et al., “Meningoencephalitis in mice infected with an equine herpesvirus 1 strain KyA recombinant expressing glycoprotein I and glycoprotein E,” Virus genes 29(1):9-17 (August 2004); C. F. Colle et al., “Equine herpesvirus-1 strain KyA, a candidate vaccine strain, reduces viral titers in mice challenged with a pathogenic strain, RacL,” Virus Research 43(2):111-124 (August 1996); T. Kondo et al., “A protective effect of epidermal powder immunization in a mouse model of equine herpesvirus-1 infection,” Virology 318(1):414-419 (January 2004); C. Walker et al., “Comparison of the pathogenesis of acute equine herpesvirus 1 (EHV-1) infection in the horse and the mouse model: a review,” Veterinary Microbiology 68(1):3-13 (August 1999); K. M. Ruitenberg et al., “DNA-mediated immunization with glycoprotein D of equine herpesvirus 1 (EHV-1) in a murine model of EHV-1 respiratory infection” Vaccine 17(3): 237-244 (February 1999); P. A. M. van Woensel et al., “A mouse model for testing the pathogenicity of equine herpes virus-1 strains,” Journal of Virological Methods 54(1):39-49 (July 1995); M. K. Baxi et al., “Molecular studies of the acute infection, latency and reactivation of equine herpesvirus-1 (EHV-1) in the mouse model,” Virus Research 40(1):33-45 (January 1996).
While the murine model has served its purpose as a model for simple EHV-1 infections characterized by clinical signs such as pyrexia (abnormally elevated body temperature) or loss of body weight, the mouse as an experimental model for studying the neurological disease and efficacious vaccine candidates in large horses has several drawbacks. First of all, the EHV-1 infection in the mouse never can progress to express the same breadth of neurological signs that plague the natural host, namely, horses, thus failing to provide a complete picture as a viral vaccine candidate model to permit adequate vaccination strategies in horses. For instance, myeloencephalopathic disease like that exhibited by herpesvirus infected horses does not develop in the experimental mouse model of herpesvirus disease. Secondly, a major adverse effect of infection in pregnant mares is the induction of spontaneous infectious abortions, which cannot be duplicated or investigated in the vaccination studies run in the classic murine model. Thirdly, without a solid understanding of the clinical signs and progression of the EHV-1 disease in horses, the vaccine candidates will not have broad applicability and activity to prevent more serious neurologic signs than simple infections caused by the single etiological agent in the mouse. More research on the progression of the disease in the natural host is necessary beyond the bare minimal effects from infection in the mouse. Fourthly, the detection of new mutant equine herpesviruses has further limited the application of the murine model as a practical animal model for vaccine purposes.
Eventually, final testing of the safety and efficacy of the virus strains will take place in vivo in horses. Equine vaccines, which are commercially available and described in the art, are subsequently tested on horses, but typically only after the initial efficacy and safety studies in the classic murine model that leave a wide gap in vaccination data. For a case directly on point, the horse vaccines comprising a mutant EHV-1 or EHV-4 virus disclosed in U.S. Pat. No. 7,060,282 are shown as being conventionally tested first through in vivo studies in laboratory mice. The patentees note the limitations of using the standard murine model to confirm protective activity in horses. They indicate that while pathogenicity of individual EHV-1 strains can be correlated from the mouse model to the behavior in the natural equine host, more conclusive proof is obtained only from vaccination trials in horses. Patentees further complain that the level of protection against challenge infection and ultimately in preventing abortion in pregnant mares can only be established in the target animal and, thus, the initial murine testing is of limited value in the final analysis.
There is a definite art-recognized problem with the classic mouse model that the present invention solves by developing the first equine disease model for the successful reproduction of herpesvirus-1 neurological disease in the horse.
It is therefore an important object of the present invention to provide a unique equine experimental disease model that is useful for evaluating and developing safe and effective vaccines in the protection of horses against equine herpesvirus infections and, more specifically, against equine herpesvirus-1 neurological disease.
It is a further important object of the present invention to identify the parameters in equine herpesvirus inoculated horses that are highly correlated with post-inoculation development of clinical disease and particularly to find the quantifiable risk factors that are directly associated with the development of clinical neurological signs in horses exposed to equine herpesvirus-1.
It is another important object of the invention to find an effective modified live, virus vaccine that protects horses against neurological disease due to infection caused by equine herpesvirus-1. While presently known EHV-1 vaccines that claim protection against neurologic disease as well as respiratory disease and abortion are largely based on inactivated viruses (see, for example, U.S. Pat. Nos. 7,323,178 and 7,226,604), it is art-recognized that live and attenuated viral vaccines, as a general rule, can induce a better immune response. Therefore, this goal of the present invention is to provide a new and improved live, attenuated vaccine formulation that stimulates a protective immune response against neurologic disease due to equine herpesvirus-1.
Further purposes and objects of the present invention will appear as the specification proceeds.
The foregoing objects are accomplished by providing a novel equine disease model, the determination of the quantifiable risk factors for the post-exposure development of equine herpesvirus-1 neurological disease and a unique equine herpesvirus-1 vaccine as described herein.